Renal metabolic response to acid base changes: II The early effects of metabolic acidosis on renal metabolism in the rat

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Renal metabolic response to acid-base

changes: II. The early effects of metabolic

acidosis on renal metabolism in the rat

George A. O. Alleyne

J Clin Invest.

1970;

49(5)

:943-951.

https://doi.org/10.1172/JCI106314

.

The early renal metabolic response was studied in rats made acidotic by oral feeding of

ammonium chloride. 2 hr after feeding of ammonium chloride there was already significant

acidosis. Urinary ammonia also increased after ammonium chloride ingestion and at 1½ hr

was significantly elevated. In vitro gluconeogenesis by renal cortical slices was increased at

2 hr and thereafter increased steadily. Ammonia production by the same slices was also

increased at 2 hr, but thereafter fell and at 6 hr had decreased to levels which, although

higher than those of the control, were lower than those obtained from the rats acidotic for

only 2 hr. There was no correlation between in vitro gluconeogenesis and ammonia

production by kidney slices from rats during the first 6 hr of acidosis, but after 48 hr of

ammonium chloride feeding, these two processes were significantly correlated. The early

increase in renal gluconeogenesis was demonstrable with both glutamine and succinate as

substrates.

The activity of the enzyme phosphoenolpyruvate carboxykinase was increased after 4-6 hr

of acidosis. During this time there was a decrease in renal RNA synthesis as shown by

decreased uptake of orotic acid-

5

H into RNA.

Metabolic intermediates were also measured in quick-frozen kidneys at varying times after

induction of acidosis. There was an immediate rise in aspartate and a fall in

a

-ketoglutarate

and malate […]

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Renal Metabolic Response to Acid-Base Changes

II. THE

EARLY

EFFECTS OF METABOLIC ACIDOSIS

ON RENAL METABOLISM IN

THE RAT

GEORGE

A.0.ALLEYNE

FromtheMedical Research Council, Tropical Metabolism Research Unit,

University oftheWestIndies, Mona, Kingston 7, Jamaica

ABSTRACT The early renal metabolic response was studied in rats made acidotic by oral feeding of ammo-nium chloride. 2 hr after feeding of ammonium chloride there was already significant acidosis. Urinary ammonia also increased after ammonium chloride ingestion and

at 1 hr was significantly elevated. In vitro gluconeo-genesis by renal cortical slices was increased at 2 hr and thereafter increased steadily. Ammonia production by the same slices was also increased at 2

hr,

but there-after fell and at 6 hr had decreased to levels which, although higher than those of the control, were lower than those obtained from the rats acidotic for only 2 hr.

There was no correlation between in vitro gluconeo-genesis and ammonia production by kidney slices from rats during the first 6 hr of acidosis, but after 48 hr of ammonium chloride feeding, these two processes were

significantly correlated. The early increase in renal gluconeogenesis was demonstrable with both glutamine

and succinate as substrates.

The activity of the enzyme phosphoenolpyruvate carboxykinase was increased after 4-6 hr of acidosis. During this time there was a decrease in renal RNA

synthesis as shown by decreased uptake of orotic

acid-3H into RNA.

Metabolic intermediates were also measured in

quick-frozenkidneys at varying times after induction of acido-sis. There was an immediate rise in aspartate and a fall in a-ketoglutarate and malate levels. There was never any difference in pyruvate or lactate levels or

lac-tate:pyruvate ratios between control and acidotic rats. Phosphoenolpyruvate rose significantly after 6 hr of acidosis.

All the data indicate that increased gluconeogenesis is an early response to metabolic acidosis and will

This work was presented in part at the 4th International Congress of Nephrology, Stockholm, Sweden, May 1969.

Receivedfor publication 10 November 1969 and in revised

form2January 1970.

facilitate ammonia production by utilization ofglutamate

which inhibits the glutaminase I enzyme. The pattern

of change in metabolic intermediates can also be

inter-preted as showing that there is not only enhanced glu-coneogenesis, but also that there may be significant increase of activity of glutaminase II as part of the very early response to metabolic acidosis.

INTRODUCTION

Urinary excretion of ammonia is essential for main-tenance of normal acid-base homeostasis in several spe-cies including man, rat, and the dog, and it was

demon-strated some 20 yr ago (1) that an increase in urinary ammonia is a typical response to metabolic acidosis. In spite of this, there is no agreement as to the early

sequence ofeventsfollowing acidosis or the fundamental nature of the stimulus to the

amnmoniagenesis

which

occurs.

Previous reports from this laboratory have described the response of the rat kidney to metabolic acidosis of 48 hr duration (2, 3). This consisted of certain changes

in metabolic intermediates and significant increases in

activity of the enzyme phosphoenolpyruvate carboxy-kinase (PEPCK). These changes were presented in the light of a close relationship between gluconeogenesis

and ammonia production which has been postulated (4, 5) andweproposedthat the control of the two processes lay at the conversion of oxaloacetate to

phosphoenol

pyruvate by the enzyme PEPCK. It was shown further that there was an increase in enzyme activity as early

as 6 hr after induction of metabolic acidosis.

The present experiments represent an attempt to

ex-amine the early events which occur in the kidney in

response to ammonium chloride-induced acidosis. By measuring key metabolic intermediates, PEPCK activity,

in vitro gluconeogenesis, and ammoniagenesis we can show a set of events which temporally are

closely

(3)

nected and perhaps indicate the way in which the rat

increases ammonia production in response to an acid

load.

The results show that urinary anmmonia increases

112

hr after NH4C1 is given orally, and renal cortical slices

taken from rats fed NH4C1 2 hr previously, already shoxv

enhanced gluconeogenesis and ammoniagenesis, but the

two processes are not as closely related as in the more chronically acidotic rats. It is suggested that activation

of the glutaminase II system is also part of the early

response to acidosis.

METHODS

Except where stated, all experiments were performed on male Sprague-Dawley or Wistar rats which weighed be-tween 175 and 250 g and were previously fed on Purina Laboratory Chow (Ralston Purina Company, St. Louis,

Mo.). There was no difference between the two strains in the metabolic responsetoacidosis.

Induction ofacidosis. The rats were all deprived of solid food for 12 hr before sacrifice and fed by stomach tube. The

experimental group received 2.5 ml/100 g body weight of a

400 mm aqueous NH4Cl solution and rats were sacrificed at 2, 4, or 6 hr later. Controls received an equal volume of

water. In the experiments in which acidosis was continued for 48 hr, the feeding protocol was as described previously

(3). In most experiments, control and acidotic rats were

sacrificed simultaneously.

Anesthetic. Sodium pentobarbital 40 mg/kg body weight

was used routinely. When blood was needed, it was with-drawn into heparinized syringes from the exposed abdomi-nal aorta.

Ammonia excretion. Four female rats (160-180 g) were anesthetized with sodium pentobarbital 20 mg/kg body

weightand the bladder was catheterized. Small quantities of

pentobarbital were given as required to maintain light

anes-thesia. Urine was collected for periods of 30 min and at

the end of the collection period the bladder was emptied by

manual compression and washed twice with 0.5 ml aliquots

of 0.9% NaCl and once with 0.5 ml of air. The urine and

washings were adjusted to a volume of 5.0 ml for measure-ment of ammonia. After two control periods the rats re-ceived by stomach tube 1.25 ml/100 g body weight of a

800 mm aqueous NH4Cl solution. Urine collection was

then continued for 4 hr.

In vitro gluconeogenesis. Slices of kidney cortex were cut by hand by the method of Deutch (6) and washed in cold 0.9% NaCl. Portions of slices which yielded 4-10 mg

dry weight were placed in 50-ml conical flasks which

con-tained 5.0 ml of Krebs-Ringer bicarbonate buffer with the appropriate substrate and which had previously been gassed

with95% 02and5% C02. The flaskswere quickly stoppered

and flushed with the same gas mixture. Incubation was

car-ried out for 1 hr at

370C

in a metabolic bath which oscil-lated at 100/min; at the end of incubation, flasks were

removed and slices taken out and placed into tared tubes for drying at 100'C for 4 hr. Glucose and ammonia were

measured in aliquots of the medium. The pH of the medium

was 7.35-7.42 and did not change significantly during incu-bation. Net glucose or net ammonia production refers to the

glucose or ammonia produced in the presence of substrate

minus glucose or ammonia produced in identically treated flasks which contained no substrate.

Orotic acid-2H injections. The uptake of orotic acid-2H

into the total RNA of kidney cortex was used as a measure of RNA synthesis. 20

IzCi

of orotic acid-2H-0.2 ml of a

solution with a specific activity of 1 Ci/mM-was given by

intraperitoneal injection 2 hr before sacrifice; therefore, in the rats acidotic for 2 hr, NH4Cl and orotic acid had to be given simultaneously.

Metabolic intermediates. Rats were anesthetized at the appropriate times and kidneys were rapidly removed and crushed in a stainless steel mortar which contained liquid

air. The powdered frozen kidney was homogenized in 6%

perchloric acid in aJencons glass homogenizer with a Teflon pestle (Jencons, Hemel Hempstead, England). The

protein-free supernatant remaining after centrifuging was

neutral-iLed with 5 M K2C03 in the standard manner (7). The protein precipitate was dissolved in 2.0 N NaOH.

Assays.-Blood pH was measured with a Radiometer micro Astrup assembly (Copenhagen, Denmark). Plasma CO2 was measured with a Natelson micro gasometer

(Sci-entific Industries Inc., Springfield, Mass.). The RNA and DNA in kidney cortex were extracted and measured by methods described by Munro and Fleck (8).

Metabolic intermediates were all measured spectrophoto-metrically by standard enzymatic methods (7) which em-ployed NADH oxidation or NAD reduction. For measure-ment of malate, the acetyl pyridine analogue of NAD was used. Pyruvate was always measured within 6 hr of sacri-fice of the animals. Phosphoenolpyruvate carboxykinase (PEPCK) was measured in kidney cortex by two methods. The decarboxylation of oxaloacetate was as described by Nordlie and Lardy (9) and the bicarbonate-"'C exchange assay wasthat of Chang and Lane (10). For the decarboxy-lation assay, slices of kidney cortex were homogenized in ice-cold 0.25 M glucose, and the resultant homogenate

cen-trifuged at 78,000 g in a Beckman model L preparative ultracentrifuge for 2 hr (Beckman Instruments, Inc., Ful-lerton, Calif.). The supernatant solution was used for the assay. For the bicarbonate exchange, the homogenate was centrifuged at approximately 5000 g for 30 min to remove the mitochondria and nuclei, and the supernatant was used. Protein was measured by the method of Lowry, Roseborough, Farr, and Randall (11). Ammonia was measured colori-metrically (12). Glucose was measured by the specific glucose oxidase method (13).

Counting of radioactivity. The RNA-2H and the

"4C-labeled malate in the bicarbonate exchange assay of PEPCK were counted in a Beckman L.S. 150 liquid scin-tillation counter with external standard channels ratio. The scintillant was 0.6% PPO (2,5-diphenyloxazole) in a toluene: Triton X (2: 1) mixture.

RESULTS

Acid-base status (Table I). Oral NH4Cl produced a

significant degree of metabolic acidosiswhich was

maxi-mal at 2 hr. (Throughout this paper, the statement that

differences between means are significant indicates a P value of at least 0.05 as determined by Student's t

test.) There was a rise in blood hydrogen ion concen-tration and a fall in plasma C02. At 4 and 6 hr after the NH4C1, the acidosis wvas less severe. Similar data

for rats acidotic for 48 hr havebeen reported previously

(3).

(4)

Ammonia excretion (Fig. 1). This shows that after an intragastric acid load, urinary ammonia started to rise and at

11

hr was already significantly higher than control values. The ammonia excretion appeared to plateau between 2 and 4 hr.

In vitro gluconeogenesis and ammoniagenesis. Rats

were sacrificed at the indicated times after induction of metabolic acidosis. With 10 mm glutamine as substrate

(Table II) there was increased gluconeogenesis after 2 hr of acidosis. There was no significant difference in gluconeogenesis in kidneys from rats sacrificed at 2 and 4 hr, but apart from this, there was a steady increase in gluconeogenic capacity and after 48 hr of acid feeding gluconeogenesis was twice the control values. For the 48-hr studies, rats were fed NH4Cl in glucose every 12 hr so renal gluconeogenesis was measured in a group of control rats fed only glucose for 48 hr. Their net glucose production was 75.5

±2.8

,umole/g dry weight per hr (mean +SEM, n=7). This value is not different from the control values in Table II which were from rats given only water once by stomach tube.

In vitro ammonia production is shown in Table III. There was a significant increase in ammonia production from rats acidotic for only 2 hr. However, in contrast with the gluconeogenesis data, there was a subsequent decrease in ammonia production, and the value for rats acidotic for 6 hr, although higher than the control value is significantly lower than that obtained in rats acidotic for 2 or 4 hr. After 48 hr of acidosis, ammonia produc-tion from cortical slices had risen again and was

sig-nificantly higher than that at 6 hr but still not higher thanthatat2hr.

An attempt was made to correlate gluconeogenesis and ammonia production from cortical slices. There was

I5

._

ob ICL

3

-S.

O_

IC

-I

0

_1 O

NHCI given

I

TABLE I

Blood Hydrogen Ion ConcentrationandPlasma CO2 in Rats

at Varying Timesafter Feeding

Time Group Blood H PlasmaC02

hr nEq/liter mmoles/liter

2 Control (19)* 43.6 40.8t 24.5 40.5 Acidotic (21) 79.04-1.9 13.8 ±0.4 4 Control (10) 40.4 41.8 26.440.8

Acidotic(11) 66.0±2.9 16.8±0.4 6 Control(19) 40.5 41.4 24.2 ±0.4

Acidotic (22) 58.1 ±1.4 16.5±0.3

* The numbersinparentheses referto the number of animals. t Mean ±-SEM.

no statistical correlation between ammonia and glucose

production for the first 6 hr, but in the rats acidotic for 48 hr, a significant correlation existed (Fig. 2).

With succinateas substrate therewasalsoan increase in gluconeogenesis in slices from the acidotic rats (Ta-ble

IV).

The values at 2, 4, and 6 hr are all higher

than control, but there is no significant rise in gluco-neogenic capacity with increasing duration of acidosis

as was seen when glutamine was used as substrate. The most probable reason for this is that 180-190

Amole/g dry weight per hr represents the maximal

glu-coneogenic capacity of the tissue. The possibility that

ammonium ions stimulated

gluconeogenesis

was

con-sidered, so cortical slices were incubated with either 10 mM succinate or 10 mM glutamine as substrate in flasks

to which NH4Cl was added to a final concentration of

0.1, 0.2, or 0.5 mmole/liter. All experiments were done

Ai~~~

IL

I

i

Mean±SEM

2 3 4

Time- hours

(5)

TABLE II

GlucoseProduction by Kidney Cortical Slices after Acidosis of Varying Duration

(Substrate10mMGlutamine)

Duration ofacidosis, hr

Control 2 4 6 48

Glucose production,

,umoles/g ofdrywtper hr 72.9* 90.9 95.9 115.5 136.5

3.6t 3.4 5.4 5.5 6.3

Significance of difference

fromcontrols, P - <0.005 <0.005 <0.001 <0.001

2 hr,P - NS <0.001 <0.001

4hr, P - <0.025 <0.001

6hr,P - - <0.025 Each value represents the mean of at least 14 observations.

* Mean.

S5EM.

at the same pH. There was no stimulation of

gluco-neogenesis by ammonium ions (Table V).

PEPCK activity. Since it was shown previously (3) that PEPCK was the key gluconeogenic enzyme stimu-lated by acidosis, this enzyme was assayed at the times indicated. Two assays were used: the oxaloacetate

de-carboxylation and the bicarbonate exchange. The latter used partly as a check on the data obtained by the

former which is technically more difficult, and also

because it is being used in this laboratory for further studies on the metabolic control of the increase in en-zyme activity resulting from acidosis. The results of

the decarboxylation assay are shown in Table VI. As

early as4 hr after making the rats acidotic, there was a

significant increase in enzyme activity. With the

ex-change assay (Table VII) there is a significant

in-crease in activity at 6 hr, and at 48 hr this increase

wasstillgreater.

Incorporation of orotic acid-8H into RNA (Table

VIII). This was measured to see whether the

in-creased enzyme activity was associated -with increased RNA synthesis. Acidosis depressed the incorporation of orotic acid-3H into kidney cortex RNA. At 2 hr this inhibition was marked and was less at 4 hr, but there was no significant difference between control and aci-dotic rats at 6 hr. The decrease in RNA synthesis shown by the decreased incorporation of orotic

acid-2H

into RNA is not reflected in the tissue RNA levels or the RNA: DNA ratios which remained normal

through-out the 6 hr period of acidosis (Table IX). This

ex-TABLE III

Ammonia ProductionbyKidneyCortical Slices after Acidosis of Varying Duration (Substrate 10mM Glutamine)

Duration ofacidosis,hr

Control 2 4 6 48

Ammoniaproduction,

/Amoles/g of drywtper hr 688.1* 949.6 929.7 799.7 1045.1

27.91

14.3 41.2 30.0 41.8

Significanceofdifference

from controls,P <0.001 <0.001 <0.02 <0.001

2 hr,P - NS <0.02 NS

4hr,P <0.025 NS

6hr,P <0.001

Eachvaluerepresentsthe mean of 9-14 observations. *Mean.

$SEM.

(6)

.

0

0 0

0 0

0 0

0

0

I

100 120

Glucose production

140

,pmokes/grom

160

w0o

dry weight/hour

FIGURE 2 The relationship between in vitro gluconeogenesis and ammonia production in cortical slices fromrats acidotic for 48 hr.r=0.84; P<0.001; regression equation isy=5.6x +280.7.

periment was not done at 48 hr, since it has already

been reported (14) that at that time there is an in-crease of renal RNA.

Levels of metabolic intermediates (Table X). Even after 48 hr of acidosis pyruvate and lactate were the same in control and acidotic rats, and there was no

significant difference in the lactate: pyruvate ratios be-tween control and acidotic rats at any stage. Both py-ruvate and lactate tended to be higher in the more

chronically acidotic rats. There was a significant de-crease in ca-ketoglutarate at 2 hr, and this was still ap-parent in the rats which were acidotic for 48 hr. The

changes in malate levels were similar to those for

a-ketoglutarate. Aspartate was significantly higher in TABLE IV

Glucose ProductionbyKidney Cortical Slices after Acidosis of Varying Duration

(Substrate 10mMSuccinate)

Duration ofacidosis,hr

Control 2 4 6

Glucoseproduction,

gmoles/g of drywtPerhr 161.2* 191.7 183.2 190.6

6.8$ 5.6 5.1 6.7

Significanceofdifference

fromcontrols, P - <0.005 <0.02 <0.01

Eachvalue represents themeanof 11-14observations.

*Meani.

1SEM.

the acidotic rats at 2, 4, and 6 hr, but by 48 hr there was no difference between the two groups of animals.

Phosphoenol pyruvate levels were unchanged at 2 and 4 hr, but by 6 hr they had risen significantly in the acidotic rat. This rise was even more striking at 48 hr.

DISCUSSION

The data show that as early as 2 hr after NH4Cl, rats

already have a severe metabolicacidosis, and in response to this, there is increasedurinary excretion of ammonia.

Rats were not sacrificed earlier than 2 hr after the acid load, but judging from the increase in

urinary

ammoniawhich is apparent

1J

hrafter ammonium

chlo-TABLE V

Effect of Varying Concentrationsof NH4+

on In VitroGluconeogenesis

Glucoseproduced

NH4Cl Substrate10mm Substrate10mM

concentration succinate glutamine mmoles/liter jimoks/gof dry wtper hr

0 150.5

±11.8*

(6)t

83.4±8.4 (4)

0.1 161.4

±9.2

(6) 88.4 411.9 (4)

0.2 157.1 46.4 (6) 83.5 ±6.6 (4) 0.5 145.4

±5.5

(6) 72.8 49.2 (4) *Mean

±SEM.

tNo. of observations.

Early Renal Response to Metabolic Acidosis 947

1400

°0

120Q

4-0

c

._

Or

2 -10

< at 800

60Q

(7)

TABLE VI

PEPCK Activity in Kidney Cortex from Rats with NH4Ct-InducedAcidosis ofDifferent Duration

(Decarboxylation Assay)

Durationof No.of

acidosis Group rats PEPCK*

hr

2 Control 6 97.7 ±12.3k

Acidotic 8 90.3 ±7.2 4 Control 6 100.5 48.7 Acidotic 9 154.6±t12.7 * PEPCK activity in nanomoles of PEP formed per minute permilligram of protein.

tMean

±SEM.

ride, there must be a significant degree of acidosis even earlier than 2 hr. The rise in urinary ammonia at

1J

hr

indicates an increase in renal ammonia production. It has been shown that glutamine is the major source of urinary ammonia, and in dogs acidotic for 3 days the amide and amino groups of glutamine contribute

35-50% and 16-25% respectively of the urine ammonia (15). There are two possible pathways by which

glu-tamine may contribute to renal ammonia. Gluglu-tamine may be deamidated to form glutamate and ammonia (en-zyme-glutaminase I) or alternatively, the amino group may be removed by transamination with the resultant

formation of a-ketoglutaramate and an amino acid. The a-ketoglutaramate is deamidated to give a-ketoglu-tarate and ammonia by a highly active enzyme w-ami-dase. The glutamine transaminase and w-amidase are

col-lectively referred to as the glutaminase II system.

TABLE VII

PEPCKActivity in Kidney Cortex from Rats with NH4Cl-InducedAcidosisofDifferentDuration

Bicarbonate 14CExchange Assay

Duration of No. of PEPCK acidosis Group rats activity*

hr

2 Control 5 55,630 i4,980t

Acidotic 5 60,258

±t17,550

4 Control 5 64,812 ±2,307

Acidotic 5 65,998 ±4,524

6 Control 15 49,731 ±4,049

Acidotic 15 65,472 44,237

48 Control 5 51,228

±2,460

Acidotic 4 114,599±7,605

* PEPCK activity in counts per minute of

14HCOj-

incor-porated into malate/100 mg of protein per 4 min of assay.

tMean

±SEM.

TABLE VIII

Incorporation ofOroticAcid-3Hin RNA ofRatKidneyCortex after DifferentDurationofAcidosis

Duration of No.of Oroticacid-'H

acidosis Group rats uptake into RNA

hr cpm/mg RNA

2 Control 7 23,759.3±1,619.4*

Acidotic 5 5,315.5

±597.0

4 Control 5 24,455.0 ±1,097.0

Acidotic 6 15,417.1 4±1,116.8 6 Control 5 31,704.8 45,798.7 Acidotic 6 21,282.8 ±3,568.4 *Mean SEM.

Enhanced gluconeogenesis in response to acidosis was considered important in that, at least in the rat, it served to remove the glutamate formed as a product

of the glutaminase I reaction since glutamate itself in-hibited the enzyme (4, 16).

Thepresent data show that increased renal

gluconeo-genesis is a very early response to metabolic acidosis,

and as such would facilitate the early increase in renal ammonia production. There is increased gluconeogenesis

in vitro from both glutamine and succinate, and the

pat-tern of metabolic intermediate suggests that there is increased gluconeogenesis in vivo. It is known that renal glutamate levels decrease rapidly with acidosis (15) and the fall in a-ketoglutarate and malate shown here are in keeipng with increased flux along the

gluco-neogenesis pathway. Levels of phosphoenolpyruvate (PEP) did not rise until 6 hr after acidosis, but since the level of any metabolic intermediate is a reflection of the activities of enzymes before and after it in a given

pathway, the normal PEP levels

4do

not preclude the supposition that there is increased gluconeogenesis in

TABLE IX

RNA and RNA /DNA Ratios in Kidney Cortex ofRats

after Acidosis of Different Duration

Duration

of No. of

acidosis Group rats RNA RNA/DNA

hr mg/g cortex

2 Control 10 5.09 ±0.18* 1.17 ±0.04 Acidotic 9 4.80±0.19 1.05 ±0.04

4 Control 10 5.18 i0.07 1.11 ±0.05

Acidotic 10 5.10±0.10 1.15 ±0.08 6 Control 10 4.93±0.26 1.10 ±0.06 Acidotic 9 5.07±0.29 1.14±0.04

*Mean ±SEM.

(8)

TABLE X

Levels ofMetabolicIntermediates in Whole Kidneys of Rats withNH4CI-InducedAcidosis of DifferentDuration

Duration ofacidosis,hr

Intermediate Group 2 4 6 48

nmoles/100mgof protein

Pyruvate Control 21.5 ±5.5* 19.5±3.1 23.1 ±3.1 35.94:5.5 Acidotic 24.1:4:2.7 19.6 42.1 27.8±2.4 33.5 ±3.6

Lactate Control 333.7451.1 270.6 ±34.3 303.9±31.5 619.8 4170.8 Acidotic 326.1 ±52.6 295.3±46.7 385.6±76.7 419.4±99.3

Lactate/Pyruvate Control 15.1 ±1.6 14.9±2.1 13.6 ±0.9 15.9 42.5 Acidotic 13.3±1.7 16.2 ±3.4 13.6±1.8 16.9 ±1.4 a-ketoglutarate Control 120.6±25.1 158.9±22.0 149.6±12.6 199.0 431.0

Acidotic 55.4 +5.4 39.8±2.1 42.1 ±4.1 84.3 48.0 Malate Control 126.2 ±16.7 110.5 ±15.6 125.0±11.1 118.5 ± 8.8 Acidotic 91.3 ±7.1 80.1

±11.0

74.8 43.8 91.9±7.7

Asparatate Control 580.9 ±48.9 623.7 ±42.5 559.5±35.0 664.6±25.3

Acidotic 793.7 ±39.1 846.8 415.1 751.6437.2 646.7±33.2

Phosphoenol pyruvate Control 33.3 ±2.0 30.2±2.3 39.9 ±2.9 37.3±2.7 Acidotic 35.8±1.1 33.1 41.7 47.9 ±2.3 60.0±4.4

Each value represents the mean of at least five rats except control lactate at 48 hr where only four ratswere used.

*Mean

±SEM.

vivo as early as 2 hr after ammonium chloride was given. Indeed, in spite of increased gluconeogenesis glucose-6-phosphate which is distal to PEP in the glu-coneogenic sequence, does not show any change even after 48hr of acidosis (Alleyneand Flores, unpublished data). This probably indicates that there is another rate-limiting enzyme between PEP and glucose-6-phos-phate. Goodman, Fuisz, and Cahill (4) reported that in vitro gluconeogenesis was not increased until 12 hr after induction of acidosis. The reason for this is not clear but it is possible that administration of ammonium chloride in dextrose may have suppressed the early

glu-coneogenic response in their animals since glucose

feeding depresses renal PEPCK activity (Alleyne,

un-published data).

It was suggested previously that PEPCK was the

con-trolling enzyme in the gluconeogenic response to acido-sis (3). Both methods of enzyme assay show early in-creases in PEPCK activity. The decarboxylation assay is the more physiologically important here since it is via the decarboxylation of oxaloacetate that gluconeo-genesis takes place. In any case both assays show an increased PEPCK activity 4-6 hr after induction of

acidosis.

RNA metabolism was investigated to see whether the increase in enzyme activity was associated with en-hanced RNA and presumably new protein synthesis. If

uptakeof orotic

acid-8H

into RNA can be equated with

RNA synthesis, it can be said that there was decreased RNA synthesis up to 4 hr after the acid load. The fact that RNA levels and RNA: DNA ratio did not change

is inkeeping with the observation that in the ratkidney hypertrophied as a result of contralateral nephrectomy

or acidosis, there is no change in renal RNA until after 24 and 36 hr (14). The depression of RNA synthesis in

the presence of enhanced enzyme activity may indicate

that there is a small separate fraction of RNA which

codes for the PEPCK protein, is not affected by the

general depression of RNA synthesis, and is

undetecta-ble since it is relatively small. Alternatively, the

in-creased enzyme activity may not denote increased en-zyme synthesis, but there may be activation of a previously inactive form of the enzyme. Such a change has been suggested for liver PEPCK after tryptophan injection (17). The rise in enzyme activity 4 hr after

NH4C1 is rapid, but not surprising since Lardy, Foster,

Shrago, andRay (18) have showna twofold increasein

liver PEPCK 3-4 hr after injection of a single dose of

hydrocortisone.

Although both gluconeogenesis and ammonia

pro-duction were increased as part of the early response to

metabolic acidosis, these two processes were apparently

not as closely linked as in themore chronically acidotic

rat. In rats acidotic for 48

hr.,

it has been shown that

PEPCK activity is well correlated with urinary

(9)

corre-lationbetween in vitro glucose and ammonia production.

2 hr after the acid load, in vitro ammonia production

rose, but then declined significantly; on the contrary

glucose production rose steadily. In addition, there was

no statistical correlation between in vitro glucose and

ammonia production in these early stages. These

obser-vations led to the speculation that it was the increased renal ammonia which was the stimulus for the increased gluconeogenesis. The experiments in which varying con-centrations of ammonium chloride were tried in vitro

did not give any support to this thesis.

It was suggested that the increased renal gluconeo-genesis evoked by metabolic acidosis was important for the removal of glutamate which, although it was the product, inhibited the glutaminase I enzyme (4). There

is possibly evidence here to show that this need not

be the major role for the early gluconeogenic response

to acidosis, and glutamine may be metabolized signifi-cantly through the glutaminase II pathway with a-keto-glutarate the product of the reaction being removed by gluconeogenesis.

Aspartate is the transamination product of oxalo-acetate, so the early rise in aspartate levels may mean

that there was an increase in oxaloacetate transamina-tion. If this were so, we might expect that a rise in aspartate would be accompanied by a fall in oxalo-acetate levels. Oxaloacetate levels in kidney are too low to be measured accurately, but it is possible to make some deductions about them from a knowledge of the levels and ratios of other intermediates. The lactate and pyruvate levels as well as thelactate pyruvate ratios are unchanged, thus, at least in the extramitochondrial com-partment where the glutaminase II system is probably located (19), malate: oxaloacetate ratios must also be unchanged (20). Since malate levels are decreased, oxaloacetate levels must be decreased similarly, so that

in the presence of the known increased glutamine me-tabolism (21), a rise in aspartate and a fall in oxalo-acetate occur. Thus it is feasible to suggest that glu-tamine may be the amino donor to oxaloacetate with resulting formation of aspartate. If this did occur there would be increased formation of the deamination

pro-duction of glutamine-a-ketoglutaramate which is readily deamidated by the w-amidase enzyme. Thus there might be increased ammonia production along the glutaminase II pathway at this stage. The assumption inherent in this

analysis is that the measured levels of aspartate and malate are a reflection of their levels in the cytoplasm, but this is not unreasonable since they both pass freely across the mitochondrial membrane. Stone and Pitts (22) have demonstrated that in the chronically acidotic

dog the glutaminase II system may be quantitatively

significant in ammonia production.

It has been suggested that the increased metabolism of glutamine is the stimulus to increased

gluconeogene-sis (21), but this is unlikely in view of the

demonstra-tion that with in vitro reducdemonstra-tion in pH there was increased gluconeogenesis with substrates other than glutamine (23). Recently also it has been shown that 3',5'-AMP may stimulate gluconeogenesis and ammonia production in vitro (24), but there are as yet no data

to suggestthat this maybe the stimulus which increases gluconeogenesis and ammonia production in acidosis. A further proposal has been that there is a change in redox state of pyridine nucleotides in the kidney, and

this may play a role in the increased ammonia

produc-tion in response to acidosis (25).

The experiments reported here do not make it clear

whether the metabolic events described are caused entirely by the hydrogen ion or whether there is some other effector stimulus or stimuli such as those

men-tioned above. It is very probable however that this approach of measuring important metabolic intermedi-ates and correlating experiments in vivo and in vitro will ultimately detect the precise stimulus for the

meta-bolic events which follow acidosis.

ACKNOWLEDGMENTS

I am grateful to ProfessorJ. C.Waterlow, Director of this

Unit, for his advice and constant encouragement. I am

especially indebtedto Dr. Hernando Flores, David Millward, Harvey Besterman, and Claudette Cousins for fruitful dis-cussions and technical help.

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Figure

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References